Engineered Janus probes modulate nucleic acid amplification to expand the dynamic range for direct detection of viral genomes in one microliter crude serum samples

Abstract

The viral genome load in diverse clinical samples varies over several orders of magnitude (e.g. 1–10⁴ copies per μL), thus a dynamic range-extended and sensitive analysis method is highly desired. However, existing well-developed nucleic acid amplification systems always suffer from either a limited dynamic range or modest sensitivity for analysis of these samples. Herein, we propose a general engineered Janus probe to modulate the thermodynamics and kinetic properties of the amplification reaction. Through rational regulation, the Janus system improves the performance by both reducing the background and enhancing the signal, expanding the operative dynamic range by 2 orders of magnitude. This proposed approach achieves a detection limit for hepatitis B virus (HBV) DNA of down to 3 copies and can be successfully applied for direct quantification of the HBV genome in one microliter crude serum samples without any pretreatment. The results are consistent with clinical diagnosis and hold considerable potential to discriminate healthy volunteers and patients at different disease stages. Whereas, following the same operation, the representative well-developed system provided serious false-negative results using such trace amounts of samples from clinically confirmed positive patients.